The ultimate expression of the genetic makeup of a cell resides in its proteins. Although protein biosynthesis is a complex process requiring numerous proteins and nucleic acids, a major determinant of its specificity resides in the initial step-the activation and transfer of an amino acid to its respective transfer RNA by an aminoacyl-tRNA synthetase. However, relatively little is known about how the amounts of these components are determined, whether protein synthesis is regulated at the level of aminoacyl-tRNA formation, and how aminoacyl-tRNA actually functions in vivo. Since aminoacyl-tRNA formation ultimately depends on the availability of functional tRNAs and functional aminoacyl-tRNA synthetases, it is a long range goal of this work to elucidate the pathways' enzymes and, regulatory factors that determine the levels and activities of these components. This includes analysis of the structure and function of nucleases involved in tRNA biosynthesis and a determination of how the mammalian translation system actually uses aminoacyl-tRNA in vivo. The studies we propose should provide essential information in these central areas of macromolecular metabolism. The specific aims of this project are: 1) elucidation of the complete maturation pathway for a mixed function transcript containing a tRNA and a mRNA; 2) identification, characterization and in vivo function of E. coli exoribonucleases and their genes; 3) elucidation of exoribonuclease active sites and specificity of substrate recognition; 4) examination of the functional role of the high molecular weight aminoacyl-tRNA synthetase complex and of its p38 component; 5) analysis of the role of the actin cytoskeleton in the organization and function of the mammalian translation system; and 6) examination of the assembly of the mammalian translation machinery. In view of the central role of proteins in all cell functions, a detailed understanding of how these components are synthesized, and of how aminoacyl-tRNA contributes to the specificity and regulation of the translation process, is of great importance. Elucidation of these aspects of protein synthesis in normal cells is a prerequisite to the complete understanding of abnormal situations which may occur under pathological conditions.